Research Paper Ecdysone oxidase and 3-dehydroecdysone-3β ...
Glycogen Synthase Kinase 3β Inhibition and βcatenin Activation Contribute to Enhanced Survival...
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Results: After initial decrease in bodyweight post RT, there was no significant difference in bodyweight among treatment groups. A significant radioprotective effect, as measured by breathing frequencies, was observed for animals treated with MnTE-2-PyP 5+ (6 mg/kg, p = 0.02 vs. RT only) and MnTnHex-PyP 5+ (0.3 mg/kg, p = 0.02 vs. RT only). This effect was confirmed by histopathology studies. Reduction in oxidative stress (8-OHdG) was also observed in the same treatment groups (MnTE-2-PyP 5+ 6 mg/kg, p \0.001 vs. RT only and MnTnHex-2-PyP 5 0.3 mg/kg, p = 0.029 vs. RT only). TGF-b was significantly lower in MnTE-2-PyP 5+ (3 and 6 mg/ kg, p = 0.002 and p \ 0.001 vs. RT only) and MnTnHex-2-PyP 5+ (0.3 mg/kg, p \ 0.001 vs. RT only) groups. VEGF was signif- icantly lower only in the MnTE-2-PyP 5+ (6 mg/kg, p \ 0.001 vs. RT only) group. CA IX and its transcription factor HIF-1 were both lower in the MnTE-2-PyP 5+ (6 mg/kg, for CA IX p = 0.013, for HIF-1 p = 0.002 vs. RT only) group and in the MnTnHex-2- PyP 5+ (0.3 mg/kg, for CA IX p = 0.005, for HIF-1 p = 0.019 vs. RT only) group. However, only a trend towards a reduction on macrophage activation (ED-1) was seen in the MnTE-2-PyP 5+ (3 and 6 mg/kg, p = 0.069 and 0.203 vs. RT only) groups. Local toxicity at the injection sites was observed in the animals receiving MnTnHex-2-PyP 5+ (1 mg/kg or higher). Conclusion: This is the first study to compare the SOD mimics MnTE-2-PyP 5+ and MnTnHex-2-PyP 5+ in a rodent model of ra- diation induced lung injury. MnTE-2-PyP 5+ at dose of 6 mg/kg and MnTnHex-2-PyP 5+ at dose of 0.3 mg/kg given daily s.c. for two weeks were able to significantly reduce RT-induced lung injury measured by functional, histopathology, and immunohistochem- istry studies. However, MnTE-2-PyP 5+ appears to be less toxic and more effective in pulmonary radioprotection. Author Disclosure: B.M. Gauter-Fleckenstein, None; I. Batinic-Haberle, None; K.C.S.A. Fleckenstein, None; Z. Vujaskovic, NIH- U19-AI-067798, B. Research Grant. 2736 Glycogen Synthase Kinase 3b Inhibition and bcatenin Activation Contribute to Enhanced Survival After Radiation in Pancreatic Cancer Cells A. Spalding 1 , R. L. Watson 1 , S. P. Zielske 1 , A. Kim 1 , G. Bommer 1 , E. R. Fearon 1 , H. Eldar-Finkelman 2 , T. S. Lawrence 1 , E. Ben-Josef 1 1 University of Michigan, Ann Arbor, MI, 2 Tel Aviv University, Ramat Aviv, Israel Purpose/Objective(s): Pancreatic cancer responds poorly to radiation. Activation of certain signaling pathways, such as the ca- nonical or bcatenin-dependent Wnt pathway, by radiation may contribute to the resistance of cells to radiation. Prior studies have shown that activation of Glycogen synthase kinase 3b (GSK3b) can induce apoptosis directly and that GSK3b can phosphorylate multiple amino-terminal serine/threonine residues in bcatenin (bcat), leading to bcat’s ubiquitination and degradation. Activation of the bcat-dependent Wnt pathway leads to inhibition of GSK3b, allowing stabilization and nuclear translocation of bcat for tran- scription of bcat/T cell factor (TCF)-regulated cellular genes. We tested the hypothesis that irradiation activates bcat-dependent Wnt signaling, thereby modulating the response of pancreatic cancer cells to radiation. Materials/Methods: We measured levels of total and phosphorylated GSK3b by Western blotting in Panc1 and BxPC3 cells after irradiation in culture and in nude mouse xenografts. We measured bcat-dependent transcription using the bcat/TCF-dependent TOPFLASH reporter construct and quantitative RT-PCR of Axin2 mRNA expression following irradiation. We assessed b-cat nuclear localization in xenografts, using immunofluorescence on paraffin-embedded sections. After assessing the baseline and post-radiation Wnt pathway activity levels, we generated pancreatic cancer cells expressing the following constructs: i) constitu- tively active S9A GSK3b; ii) kinase inactive KK85,86MA GSK3b; iii) 3) shRNA designed to silence GSK3b; iv) constitutively active S33Y bcat, and v) empty vector controls. We performed colony formation assays to determine the ability of each mutation to influence survival after radiation, and analyzed their effect on Wnt signaling after radiation. Results: Radiation induced the phosphorylation of ser9 of GSK3b in a dose and time dependent manner in culture. One 2 Gy frac- tion induced a five-fold increase in bcat reporter activity in culture. After five 2 Gy fractions, we detected increased levels of ser9 GSK3b phosphorylation and increased nuclear localization of bcat in pancreatic cancer xenografts. Stable expression of either KK85,86MA GSK3b or S33Y bcat reduced the cytotoxicity of radiation in pancreatic cancer cells by 30 ± 5% and 25 ± 3% respectively (p \ 0.05 vs control). Conclusions: Radiation induces Wnt pathway activation as evidenced by suppression of GSK3b activity and activation of bcat. These mechanisms may in part underlie the radioresistance clinically evident in pancreatic cancers. Agents which inhibit Wnt path- way signaling may enhance the effect of radiation in pancreatic cancer. Author Disclosure: A. Spalding, None; R.L. Watson, None; S.P. Zielske, None; A. Kim, None; G. Bommer, None; E.R. Fearon, None; H. Eldar-Finkelman, None; T.S. Lawrence, None; E. Ben-Josef, None. 2737 Effect of X-Irradiation on Dendritic Spines Morphology of Hippocampal Neurons T. Nakano, K. Shirai, T. Mizui, Y. Suzuki, M. Okamoto, Y. Yoshida, W. Al-Jahdari, K. Hanamura, T. Shirao Gunma University Graduate School of Medicine, Maebashi Gunma, Japan Purpose/Objective(s): Brain irradiation is often performed in patients with brain tumors, however, irradiation sometimes leads to brain dysfunctions. We previously reported that 30 Gy X-irradiation did not induce the neural apoptotic death in mature neurons. In the present study, we analyzed whether X-irradiation cause any change on neural spines morphology in mature neurons by assess- ing dendritic spine-resident proteins drebrin and Synapsin I, immunocytochemically. Materials/Methods: We prepared primary neuronal cultures the hippocampi of fetal rats with Banker Methods. X-irradiation (30 Gy) was performed on the mature neurons at 21 days in vitro (DIV). After X-irradiation, the cells were immediately fixed with 4% paraformaldehyde and immunostained with antibody against to Synapsin I (a presynaptic marker) and drebrin (a postsynaptic actin- cytoskeleton regulator protein). Then the clusters of drebrin and Synapsin I were analyzed by MetaMorph software (Universal Im- aging, West Chester, PA). Results: Thirty dendrites in each experiment were analyzed. The number of drebrin clusters along dendrites was 97.66 ± 6.80/100 mm after 30 Gy X-irradiation, while the number of the control was 124.16 ± 6.12/100 mm. X-irradiation decreased significantly the S608 I. J. Radiation Oncology d Biology d Physics Volume 69, Number 3, Supplement, 2007
Transcript of Glycogen Synthase Kinase 3β Inhibition and βcatenin Activation Contribute to Enhanced Survival...
S608 I. J. Radiation Oncology d Biology d Physics Volume 69, Number 3, Supplement, 2007
Results: After initial decrease in bodyweight post RT, there was no significant difference in bodyweight among treatment groups.A significant radioprotective effect, as measured by breathing frequencies, was observed for animals treated with MnTE-2-PyP5+ (6mg/kg, p = 0.02 vs. RT only) and MnTnHex-PyP5+ (0.3 mg/kg, p = 0.02 vs. RT only). This effect was confirmed by histopathologystudies.
Reduction in oxidative stress (8-OHdG) was also observed in the same treatment groups (MnTE-2-PyP5+ 6 mg/kg, p\0.001 vs.RT only and MnTnHex-2-PyP5 0.3 mg/kg, p = 0.029 vs. RT only). TGF-b was significantly lower in MnTE-2-PyP5+ (3 and 6 mg/kg, p = 0.002 and p \0.001 vs. RT only) and MnTnHex-2-PyP5+ (0.3 mg/kg, p \0.001 vs. RT only) groups. VEGF was signif-icantly lower only in the MnTE-2-PyP5+ (6 mg/kg, p \ 0.001 vs. RT only) group. CA IX and its transcription factor HIF-1 wereboth lower in the MnTE-2-PyP5+ (6 mg/kg, for CA IX p = 0.013, for HIF-1 p = 0.002 vs. RT only) group and in the MnTnHex-2-PyP5+ (0.3 mg/kg, for CA IX p = 0.005, for HIF-1 p = 0.019 vs. RT only) group. However, only a trend towards a reduction onmacrophage activation (ED-1) was seen in the MnTE-2-PyP5+ (3 and 6 mg/kg, p = 0.069 and 0.203 vs. RT only) groups. Localtoxicity at the injection sites was observed in the animals receiving MnTnHex-2-PyP5+ (1 mg/kg or higher).
Conclusion: This is the first study to compare the SOD mimics MnTE-2-PyP5+ and MnTnHex-2-PyP5+ in a rodent model of ra-diation induced lung injury. MnTE-2-PyP5+ at dose of 6 mg/kg and MnTnHex-2-PyP5+ at dose of 0.3 mg/kg given daily s.c. for twoweeks were able to significantly reduce RT-induced lung injury measured by functional, histopathology, and immunohistochem-istry studies. However, MnTE-2-PyP5+ appears to be less toxic and more effective in pulmonary radioprotection.
Author Disclosure: B.M. Gauter-Fleckenstein, None; I. Batinic-Haberle, None; K.C.S.A. Fleckenstein, None; Z. Vujaskovic, NIH-U19-AI-067798, B. Research Grant.
2736 Glycogen Synthase Kinase 3b Inhibition and bcatenin Activation Contribute to Enhanced Survival After
Radiation in Pancreatic Cancer CellsA. Spalding1, R. L. Watson1, S. P. Zielske1, A. Kim1, G. Bommer1, E. R. Fearon1, H. Eldar-Finkelman2, T. S. Lawrence1,E. Ben-Josef1
1University of Michigan, Ann Arbor, MI, 2Tel Aviv University, Ramat Aviv, Israel
Purpose/Objective(s): Pancreatic cancer responds poorly to radiation. Activation of certain signaling pathways, such as the ca-nonical or bcatenin-dependent Wnt pathway, by radiation may contribute to the resistance of cells to radiation. Prior studies haveshown that activation of Glycogen synthase kinase 3b (GSK3b) can induce apoptosis directly and that GSK3b can phosphorylatemultiple amino-terminal serine/threonine residues in bcatenin (bcat), leading to bcat’s ubiquitination and degradation. Activationof the bcat-dependent Wnt pathway leads to inhibition of GSK3b, allowing stabilization and nuclear translocation of bcat for tran-scription of bcat/T cell factor (TCF)-regulated cellular genes. We tested the hypothesis that irradiation activates bcat-dependentWnt signaling, thereby modulating the response of pancreatic cancer cells to radiation.
Materials/Methods: We measured levels of total and phosphorylated GSK3b by Western blotting in Panc1 and BxPC3 cells afterirradiation in culture and in nude mouse xenografts. We measured bcat-dependent transcription using the bcat/TCF-dependentTOPFLASH reporter construct and quantitative RT-PCR of Axin2 mRNA expression following irradiation. We assessed b-catnuclear localization in xenografts, using immunofluorescence on paraffin-embedded sections. After assessing the baseline andpost-radiation Wnt pathway activity levels, we generated pancreatic cancer cells expressing the following constructs: i) constitu-tively active S9A GSK3b; ii) kinase inactive KK85,86MA GSK3b; iii) 3) shRNA designed to silence GSK3b; iv) constitutivelyactive S33Y bcat, and v) empty vector controls. We performed colony formation assays to determine the ability of each mutation toinfluence survival after radiation, and analyzed their effect on Wnt signaling after radiation.
Results: Radiation induced the phosphorylation of ser9 of GSK3b in a dose and time dependent manner in culture. One 2 Gy frac-tion induced a five-fold increase in bcat reporter activity in culture. After five 2 Gy fractions, we detected increased levels of ser9GSK3b phosphorylation and increased nuclear localization of bcat in pancreatic cancer xenografts. Stable expression of eitherKK85,86MA GSK3b or S33Y bcat reduced the cytotoxicity of radiation in pancreatic cancer cells by 30 ± 5% and 25 ± 3%respectively (p \ 0.05 vs control).
Conclusions: Radiation induces Wnt pathway activation as evidenced by suppression of GSK3b activity and activation of bcat.These mechanisms may in part underlie the radioresistance clinically evident in pancreatic cancers. Agents which inhibit Wnt path-way signaling may enhance the effect of radiation in pancreatic cancer.
Author Disclosure: A. Spalding, None; R.L. Watson, None; S.P. Zielske, None; A. Kim, None; G. Bommer, None; E.R. Fearon,None; H. Eldar-Finkelman, None; T.S. Lawrence, None; E. Ben-Josef, None.
2737 Effect of X-Irradiation on Dendritic Spines Morphology of Hippocampal Neurons
T. Nakano, K. Shirai, T. Mizui, Y. Suzuki, M. Okamoto, Y. Yoshida, W. Al-Jahdari, K. Hanamura, T. Shirao
Gunma University Graduate School of Medicine, Maebashi Gunma, Japan
Purpose/Objective(s): Brain irradiation is often performed in patients with brain tumors, however, irradiation sometimes leads tobrain dysfunctions. We previously reported that 30 Gy X-irradiation did not induce the neural apoptotic death in mature neurons. Inthe present study, we analyzed whether X-irradiation cause any change on neural spines morphology in mature neurons by assess-ing dendritic spine-resident proteins drebrin and Synapsin I, immunocytochemically.
Materials/Methods: We prepared primary neuronal cultures the hippocampi of fetal rats with Banker Methods. X-irradiation (30Gy) was performed on the mature neurons at 21 days in vitro (DIV). After X-irradiation, the cells were immediately fixed with 4%paraformaldehyde and immunostained with antibody against to Synapsin I (a presynaptic marker) and drebrin (a postsynaptic actin-cytoskeleton regulator protein). Then the clusters of drebrin and Synapsin I were analyzed by MetaMorph software (Universal Im-aging, West Chester, PA).
Results: Thirty dendrites in each experiment were analyzed. The number of drebrin clusters along dendrites was 97.66 ± 6.80/100mm after 30 Gy X-irradiation, while the number of the control was 124.16 ± 6.12/100 mm. X-irradiation decreased significantly the
